Development of an efficient and cost-effective doubled haploid production system in flax (Linum usitatissimum L.) is a prerequisite for applying doubled haploid technology to practical breeding purposes. Successful regeneration of haploid/doubled haploid plants through anther culture has been previously achieved in fiber flax and oil flax (Sun and Fu, 1981, Acta Genet Sin 8:369-374; Nichterlein et al., 1991, Euphytica 58:157-164). However, the overall efficiency of regeneration from anther culture was very low and the frequency of regeneration from the somatic tissue-derived plants was quite high (Friedt et al., 1995, Plant Breed 114: 322-326). Consequently, the efficiency of doubled haploid production was too low for any meaningful practical applications or even basic research purposes. The overall efficiency of regeneration from anther culture in flax has subsequently been improved but in these experiments the frequency of regeneration from somatic tissues remained high (Chen et al., 1998, Euphytica 102: 183-189; Chen et al., 1998, Plant Breed 117: 463-467; Chen et al., 1998, Plant Cell Reports 18:44-48). As will be apparent to one knowledgeable in the art, the progeny are therefore not all doubled haploids and must be screened for some applications. For success, it is necessary to increase the overall efficiency of regeneration, to decrease the frequency of regeneration from somatic tissues and to increase the overall efficiency of doubled haploid production.
U.S. Pat. No. 5,929,300 teaches a pollen-based transformation method wherein pollen is germinated and transformed with Agrobacterium. The treated pollen can then be used to pollinate a receptive plant. It is of note that this patent also remarks that “the cells of some plant species are not easily maintained in tissue culture and are not easily regenerated into somatic clones” (column 1, lines 31-33).
Furthermore, Dunwell and Thurling (Dunwell and Thurling, 1985, J Exp Botany 36: 1478-1491) taught that “substantially better microspore viability is achieved if anthers of both spring and winter cultivars of rape are cultured on sucrose concentrations of 16-20% rather than the more usually recommended 8-10%. These high concentrations allow embryo induction in a larger number of anthers and reduce the inter-cultivar variations in response.” Furthermore, it was noted that anthers maintained on these high concentrations did not produce macroscopic embryos and it was recommended that transfer to lower sucrose concentrations take place during the culture phase to take full advantage of the initial high survival values. However, they also noted that “perhaps the problem of secondary embryogenesis which is so frequently found amongst microspore-derived embryos of Brassica species may be caused by trauma of an approximate ten fold reduction in osmotic pressure”. Thus, the paper concludes that subsequent growth on low sucrose may not be desirable. In addition, two other papers describe the advantages of transferring microspores from high sucrose concentration to lower sucrose concentration for embryo induction in Brassica (Baillie et al. 1992 Plant Cell Reports 11:234-237; Ferrie et al. 1995 Plant Cell Reports 14:580-584). However, it is important to note that none of these papers discuss the effect of the transfer from high sucrose to low sucrose on plant regeneration (hapoid or doubled haploid plants) or the application of the protocol for transformation purposes. It is also of note that reduction of regeneration from somatic tissue was not discussed or disclosed.
Chen et al. (Chen et al., 1998, Plant Breeding 117: 463-467) teaches a high frequency method of plant regeneration from anther culture in flax by optimizing induction media composition. Therein, it is noted that “preliminary results . . . showed that culture of anthers on a medium containing 15% sucrose for a certain period of time and then transfer of anthers to a medium containing a lower sucrose concentration dramatically increased the overall efficiency of regeneration.” It is important to note that the necessary period of time and the lower sucrose concentration are not specifically disclosed.
The establishment of an efficient plant regeneration method is a prerequisite for the development of efficient transformation protocols using tissue culture. Somatic diploid tissues, e.g. hypocotyl segments or cotyledons have been used as the ex-plants to regenerate fertile transgenic flax plants through Agrobacterium mediated or particle bombardment-based approaches (Basiran et al. 1987, Plant Cell Reports 6:396-399; Zhan et al., 1988, Plant Molecular Biology 11:551-559; Wijayanto and McHughen, 1999, In Vitro Cell Dev Biol-Plant 35:456-465). However, the transformation efficiency using hypocotyl segments as ex-plants was quite low and the escape frequency was very high (Dong and McHughen, 1993, Plant Sci. 88:61-71; Wijayanto and McHughen 1999). As will be apparent to one knowledgeable in the art, protocols which increase transformation efficiency, reduce the frequency of escape and allow the regeneration of homozygous transgene lines in the T0 generation would facilitate the use of genetic transformation to improve the agronomic and quality traits of flax in order to better meet market needs. In addition, the development of a high throughput transformation protocol would facilitate the use of flax as a model species for gene discovery and functional genomics as flax has the smallest genome size of any major field crop.
Microspores and microspore-derived haploid cells (embryos/calluses) are ideal targets for genetic transformation since transgenes can be immediately fixed upon spontaneous chromosome doubling or colchicine treatment. The immediate homozygosity of transgenes in microspore-derived transgenic plants greatly simplifies the procedure for genetic analysis and isolation of homozygous transgenic lines for further applications. Successful recovery of transgenic plants through microinjection, particle bombardment, or silicon carbide whisker treatment of microspores/microspore-derived embryo/callus has been reported in a few species (Brisibe et al., 2000, J Exp Botany 51: 187-196; Neuhas et al, 1987, Theor Appl Genet 75: 30-36; Jahne et al. 1994, Theor Appl Genet 89:525-533; Stöger et al. 1995, Plant Cell Rep 14:273-278; Fukuoka et al. 1998, Plant Cell Rep 17:323-328). Agrobacterium mediated transformation of microspore/microspore-derived embryo has been reported in Brassica napus, Datura and Nicotiana (Sangwan et al., 1993, Plant Sci 95: 99-115; Swanson and Erickson 1989, Theor Appl Genet 78:831-835; Pechan 1989, Plant Cell Rep 8:387-390; Huang 1992, In Vitro Cell Dev Biol 28P:53-58). However, as discussed above, these authors did not present detailed data and the transformation efficiency was very low in these studies. Microspore-derived embryos should have similar regeneration capacity as immature zygotic embryos. One of the main advantages of microspore-derived embryos as the ex-plants for genetic transformation is the immediate isolation of homozygous transgene lines. But the advantage of using microspore-derived embryos may not be easily realized in species where a high frequency microspore embryogenesis system is not available or access to immature zygotic embryos is very convenient, such as barley, wheat, corn and rice. It is also true to species where access to other highly regenerable plant tissues such as cotyledons or hypocotyls is very convenient, such as Brassica. This explains why there are a few preliminary reports in canola using microspore-derived embryos as the ex-plants for genetic transformation and no reports of using anther culture-derived callus as the ex-plants for genetic transformation. In flax and species that do not have other highly regenerable tissues, anther culture-derived callus/embryos would be the best choice as the ex-plants for transformation.